This article is available online at http://www.jlr.org However, due to the presence of a highly potent toxin (ricin), this native oilseed plant is not considered an ideal source for hydroxy fatty acid production. Therefore, tremendous effort has recently been made in identifying genes involved in the biosynthesis of this fatty acid and using the genes to engineer oilseed crops for producing ricinoleic acid ( 3-7 ). However, to date these attempts have met with only partial success. When a hydroxylase gene from a native plant species was introduced into oilseed crops, the hydroxy fatty acid content in transgenic seeds rarely exceeded 20% of the total fatty acids ( 8-10 ). In 2008, a new hydroxylase for the biosynthesis of ricinoleic acid was identifi ed from a nonplant origin, and expression of this gene in Arabidopsis resulted in accumulation of a slightly higher level of hydroxy fatty acids in transgenic seeds ( 11 ). Very recently, coexpression of genes encoding factors involved in the networks of the hydroxylation or acyl traffi cking process along with a hydroxylase has also been attempted ( 4-7 ). Although the improved production of hydroxy fatty acids is observed, the amount in transgenic seeds still rarely exceeds 25% of the total fatty acids. It appears that selection of genes from diverse sources and addition of cofactors can make a difference for transgenic production of hydroxyl fatty acids, but production of a single hydroxy fatty acid at the commercially viable level in plants still remains as a challenging task.Microorganisms have recently emerged as promising systems for producing biofuel for transportation and platform chemicals for biopolymers because some microbes offer high output of biomass with good oil content in a short period of time and are easily used for genetic manipulation to implement a metabolic engineering strategy. Exploitation of metabolic engineering of lipid pathways in Abstract Ricinoleic acid (12-hydroxyoctadec-cis -9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system ( Pichia pastoris ) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea , was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the ⌬ 12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic ...

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Pig peripheral blood-derived mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) stimulated with mitogens ex vivo can show significant animal-to-animal variation lead to difficulty in interpreting responses in an out-bred animal species. Mixed-cell populations were stimulated ex vivo with 2.5 μg/ml Con A or 2.5 ng/ml PMA plus 250 ng/ml ionomycin (PMAi; (LPCMs only)) or media alone for 72 hours. Supernatants were then tested for cytokine production using a Bioplex assay for porcine IFNα, IFNγ, IL-10, and IL-12. Unstimulated PBMCs had significant levels of IL-10 and the median value for this group decreased in the presence of Con A. Con A did, however, induce production of IFNα and IFNγ, but not IL-12 in this cell population. In contrast, unstimulated and Con A-stimulated LPMCs produced negligible IL-10, IFNα, IFNγ, and the majority of animals' LPMCs showed negligible IL-12 production in response to Con A. In contrast, LPMCs stimulated with PMAi produced IFNγ suggesting cytokine production is mitogen-specific response. When we tracked animal-specific responses, we observed that discrete subsets of animal's PBMCs responded to Con A with significantly increased or decreased IL-10 production relative to unstimulated cells. Further, in the LPMCs, some cells produced no IL-12 in response to Con A but showed augmented production in response to PMAi, while others showed production of IL-12 in response to Con A but no response to PMAi. Flow cytometric analysis showed that the PBMCs were a mixture of CD3+ T cells>CD21+ B cells>CD172+ myeloid cells whereas the LPMCs consisted of mainly Cytotoxic T cells and Natural Killer cells. The percentage of CD8α+CD4+ antigen-experienced T cells was greater in the LPMCs relative to the PBMCs. As expected in an out-bred species, animal-specific differences in cytokine production in response to stimulants exist and may confound interpretation of results unless tracked individually.